Recombinant trypsin-like protease

ABSTRACT

PCT No. PCT/DK94/00177 Sec. 371 Date Nov. 3, 1995 Sec. 102(e) Date Nov. 3, 1995 PCT Filed May 4, 1994 PCT Pub. No. WO94/25583 PCT Pub. Date Nov. 10, 1994An active recombinant trypsin-like protease enzyme comprising the amino acid residues 25-224 of the amino acid sequence of SEQ ID NO:2, as well as a DNA construct encoding the enzyme and comprising the sequence of SEQ ID NO:1. The enzyme may be used as a detergent enzyme.

FIELD OF THE INVENTION

The present invention relates to an active recombinant trypsin-likeprotease, a proteolytic composition comprising said trypsin-likeprotease, a DNA construct comprising a DNA sequence encoding saidtrypsin-like protease, and a vector and cell harbouring the DNAconstruct. Furthermore, the present invention relates to a method ofpreparing the trypsin-like protease by use of recombinant DNAtechniques.

BACKGROUND OF THE INVENTION

Proteases are widely used as ingredients in commercial detergents toimprove the detergency towards proteinaceous soiling. For a number ofyears proteases with a broad specificity, such as subtilisins (Bacillusserine proteases) have been the most widely used detergent proteases.However, in recent years proteases having a more narrow specificity havebeen of increasing interest for detergent purposes.

WO 89/06270 discloses a detergent composition comprising a protease witha narrow substrate specificity, namely a trypsin-like protease capableof cleaving peptide bonds at the C-terminal side of lysine or arginine.The trypsin-like protease disclosed in said reference is produced byconventional fermentation of a strain of the Fusarium sp. F. oxysporumDSM 2672, and is inevitably produced together with another protease(Protease I), the existence of which is undesirable in detergentcompositions. Thus, production of the trypsin-like protease from the F.oxysporum strain involves a step in which the undesired protease isseparated from the trypsin-like protease, which makes the large scaleproduction of the latter protease more expensive than what is desirable.

It would be desirable to facilitate the production of said Fusariumtrypsin-like protease, in particular by avoiding the co-production ofthe above mentioned undesired Protease I, to be able of producing bothlarger amounts of the enzyme and to produce it in a more economicalmanner than what is possible by the prior art methods.

BRIEF DISCLOSURE OF THE INVENTION

The present inventors have now succeeded in cloning a DNA sequenceencoding a Fusarium trypsin-like protease and in obtaining expression ofan active trypsin-like protease from said DNA sequence.

Accordingly, in a first aspect the present invention relates to anactive recombinant trypsin-like protease comprising the amino acidresidues 1-224 of the amino acid sequence shown in the appended SEQ IDNo. 2.

In the present context the term "trypsin-like protease" is intended toindicate an enzyme having an activity similar to that of trypsin, i.e.an enzyme capable of cleaving peptide bonds at the C-terminal side oflysine or arginine, The trypsin-like protease activity may be determinedin an assay based on cleavage of a trypsin substrate such asN-Benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA or L-BAPNA),e.g. as described in the Materials and Methods section below.

The term "recombinant" as used about the trypsin-like protease of theinvention is intended to indicate that it is produced by a celltransformed with a DNA sequence encoding the protease. Thus, therecombinant trypsin-like protease is produced by another organism thanits parent organism and accordingly essentially free from componentsderived from said parent organism, i.e. components produced by the F.oxysporum strain DSM 2672. Such components may confer undesirableproperties to the protease, for instance by giving rise to undesirableenzymatic activities. In this respect, the recombinant trypsin-likeprotease of the invention is easily produced without Protease I, and nocostly separation similar to that performed according to WO 89/06270 isrequired.

In the course of the research leading to the present invention, which isdescribed in detail in the following examples, it was quite unexpectedlyfound to be difficult to obtain any substantial production of an activetrypsin-like protease by cultivation of a conventionally used productionorganism (Aspergillus oryzae) transformed with the DNA sequence shown inSEQ ID No. 1. On the basis of analyses of the DNA sequence and theexpression product, it was found that the enzyme encoded by the DNAsequence was expressed as an inactive proenzyme, and that large amountsof this proenzyme were expressed. Thus, even though large amounts of theprecursor form of the active enzyme were available no substantial yieldof an active maturated enzyme could be obtained, and it was concludedthat the proenzyme was unstable in the A. oryzae fermentation broth.

It was surprisingly found that the yield of the active recombinanttrypsin-like protease could be substantially increased if the medium, inwhich a host cell harbouring a DNA sequence encoding a proenzyme form ofthe enzyme was cultivated, also comprised a proteolytic enzyme capableof converting the proenzyme into an active enzyme in situ. It isexpected that the low enzymatic activity expressed when no otherproteolytic enzyme is present may be ascribed to the fact that theinactive proenzyme form of the trypsin-like protease is considerablyunstable and degraded rapidly by proteolytic enzymes expressed from thehost organism, and that the addition of a proteolytic enzyme, which morereadily than proenzyme-degrading proteolytic enzymes, reacts with theproenzyme resulting in the production of a mature, active trypsin-likeprotease.

In a further aspect the present invention relates to a DNA constructcomprising a DNA sequence encoding an active recombinant trypsin-likeprotease of the invention as defined above.

In still further aspects the present invention relates to a recombinantexpression vector harbouring the DNA construct of the invention, to acell which either harbours the DNA construct or the expression vector ofthe invention, and to a process for the production of a trypsin-likeprotease of the invention, wherein a cell of the invention as describedabove is cultured under conditions conducive to the production of theprotease, and the protease is subsequently recovered from the culture.

Finally, the present invention relates to a detergent additive anddetergent composition comprising the active trypsin-like protease of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The amino acid sequence of the recombinant trypsin-like protease of theinvention, which was isolated from a strain of the Fusarium oxysporumDSM 2672, has been aligned with that of trypsin-like proteases of otherorigins and have been shown to have a degree of homology of about 43%with that of bovine trypsin (GenBank Acc. No. P00760) and 44% with thatof Streptomyces griseus (GenBank Acc. No. P00776). The highest homologyof the trypsin-like protease of the invention has been observed withthat of the fruit fly (GenBank Acc. No. A23493), namely 48%.

These homologies are taken to indicate that some kind of evolutionaryrelationship exists between trypsin-like proteases, but also that theFusarium trypsin-like protease of the invention may represent a distinctclass of trypsin-like proteases. It is contemplated that thetrypsin-like protease of the invention or DNA encoding the protease maybe isolated from other organisms, including animals, especially amammal, an insect, a plant or a microorganism. In the present context,especially interesting origins are bacteria and fungi, including yeastsand filamentous fungi.

The active recombinant trypsin-like protease of the invention comprisingthe amino acid residues 1-224 of the SEQ ID No. 2 has a number ofcharacteristic properties. For instance, the protease surprisingly showsa reversed Arg/Lys specificity compared to that of bovine trypsin, i.e.the trypsin-like protease of the invention is more Arg-active thanLys-active.

The pH-activity profile of the trypsin-like protease of the inventionshows a broad activity optimum between pH 8 and 11, in particular about10 when using H-D-Val-Leu-Lys-pNA as a substrate at 25° C. Thetemperature optimum is approximately 40° C. at pH 9.5 usingH-D-Val-Leu-Lys-pNA. The mature, active enzyme consists of 224 aminoacid residues and has a molecular weight of 22190. It is expressed inthe form of a pre-pro-enzyme, in which the signal peptide iscontemplated to be amino acid residues -24 to -8 (according to the rulesof von Heijne (1986) and the pro-peptide amino acid residues -7 to -1.The specific activity of the enzyme is at least about 30 Casein ProteaseUnits (CPU)/g and preferably equal to or higher than about 35 CPU/g. Theprotease activity may be determined by the method given in WO 89/06270,the content of which is hereby incorporated by reference.

The DNA sequence of the DNA construct of the invention encoding arecombinant protease enzyme as defined above is preferably as shown inthe appended SEQ ID No. 1. Analogues of said sequence, which differ inone or more codons, but which encodes the recombinant protease proteinare also within the invention.

The DNA sequence of the DNA construct of the invention may be isolatedby well-known methods. Thus, the DNA sequence may, for instance, beisolated by establishing a cDNA or genomic library from an organismexpected to harbour the sequence, and screening for positive clones byconventional procedures. Examples of such procedures are hybridizationto oligonucleotide probes synthesized on the basis of the full aminoacid sequence shown in SEQ ID No. 2 or a subsequence thereof inaccordance with standard techniques (cf. Sambrook et al., 1989), and/orselection for clones expressing a trypsin-like protease activity asdefined above, and/or selection for clones producing a protein which isreactive with an antibody raised against the trypsin-like proteasecomprising the amino acid sequence shown in SEQ ID No. 2 and inparticular amino acid residues 1-224 thereof.

A preferred method of isolating a DNA construct of the invention from acDNA or genomic library is by use of polymerase chain reaction (PCR)using degenerate oligonucleotide probes prepared on the basis of theamino acid sequence of the trypsin-like protease of the inventioncomprising amino acid residues 1-224 of SEQ ID No. 2. For instance, thePCR may be carried out using the techniques described in U.S. Pat. No.4,683,202 or by R. K. Saiki et al. (1988).

Alternatively, the DNA sequence of the DNA construct of the inventionmay be prepared synthetically by established standard methods, e.g. thephosphoamidite method described by Beaucage and Caruthers (1981), or themethod described by Matthes et al. (1984). According to thephosphoamidite method, oligonucleotides are synthesized, e.g. in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

Finally, the DNA construct may be of mixed genomic and synthetic, mixedsynthetic and cDNA or mixed genomic and cDNA origin prepared by ligatingfragments of synthetic, genomic or cDNA origin (as appropriate), thefragments corresponding to various parts of the entire recombinant DNAmolecule, in accordance with standard techniques.

The recombinant expression vector carrying the DNA construct of theinvention may be any vector which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid, a bacteriophage or anextrachromosomal element, minichromosome or an artificial chromosome.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA construct of the invention, especially in abacterial host, are the promoter of the lac operon of E. coli, theStreptomyces coelicolor agarase gene dagA promoters, the promoters ofthe Bacillus licheniformis α-amylase gene (amyL), the promoters of theBacillus stearothermophilus maltogenic amylase gene (amyM), thepromoters of the Bacillus Amyloliquefaciens α-amylase (amyQ), thepromoters of the Bacillus subtilis xylA and xylB genes etc. Fortranscription in a fungal host, examples of useful promoters are thosederived from the gene encoding A. oryzae TAKA amylase, Rhizomucor mieheiaspartic proteinase, A. niger neutral α-amylase, A. niger acid stableα-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzaealkaline protease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase,

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the recombinant proteaseof the invention. Termination and polyadenylation sequences may suitablybe derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and plJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Examples of Aspergillus selection markersinclude amdS, argB, niaD and sC, a marker giving rise to hygromycinresistance. Furthermore, the selection may be accomplished byco-transformation, e.g. as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g. when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. As mentioned above thetrypsin-like protease of the invention comprising the amino acidsequence shown in the SEQ ID No. 2 comprises a preregion permittingsecretion of the expressed protease into the culture medium. Ifdesirable, this preregion may be substituted with a different preregionor signal sequence, convenient accomplished by substitution of the DNAsequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the invention, thepromoter, terminator and other elements, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al. (1989)).

The cell of the invention either comprising a DNA construct or anexpression vector of the invention as defined above is advantageouslyused as a host cell in the recombinant production of a polypeptide ofthe invention. The cell may be transformed with the DNA construct of theinvention, conveniently by integrating the DNA construct in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g. abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are grampositive bacteria such as Bacillussubtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis,Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may for instance be effected byprotoplast transformation or by using competent cells in a manner knownper se.

The yeast organism may favourably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g. Aspergillus oryzae or Aspergillus niger.Alternatively, a strain of a Fusarium species, e.g. F. oxysporum, can beused as a host cell. Fungal cells may be transformed by a processinvolving protoplast formation and transformation of the protoplastsfollowed by regeneration of the cell wall in a manner known per se. Asuitable procedure for transformation of Aspergillus host cells isdescribed in EP 238 023. A suitable method of transforming Fusariumspecies is described by Malardier et al., 1989.

In a yet further aspect, the present invention relates to a method ofproducing a recombinant trypsin-like protease of the invention, whichmethod comprises cultivating a host cell as described above underconditions conducive to the production of the protease and recoveringthe protease from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the protease of the invention. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection).

From the above disclosure it is apparent that when the trypsin-likeprotease of the invention is expressed as a proenzyme, substantiallyincreased yields may be obtained when the cultivation is performed inthe presence of a proteolytic enzyme or optionally two or moreproteolytic enzymes capable of transforming the proenzyme into an activeenzyme in the medium.

The proteolytic enzyme(s) may be added as such to the fermentation brothin which the cell producing the proenzyme is cultured. The proteolyticenzyme(s) to be added may be a Bacillus metallo-protease, for instance acommercially available enzyme, e.g. Neutrase®, thermolysin, or anotherenzyme with a similar activity such as a proteolytic enzyme fromBacillus stearothermophilus (Zamost et al., 1990) or an asparticprotease from F. oxysporum obtainable as described in Example 1hereinafter. Alternatively, the proteolytic enzyme may be produced byculturing a host cell transformed with a DNA sequence encoding theproteolytic enzyme under suitable conditions to produce the enzyme andrecovering the enzyme from the culture.

Furthermore, the presence of the proteolytic enzyme(s) in thefermentation broth may be accomplished by inserting the DNA sequence(s)encoding said enzyme(s) into the cell harbouring a DNA construct of theinvention in such a manner that the proteolytic enzyme(s) is/areexpressed and excreted in sufficient amounts to activate the protease ofthe invention.

The resulting trypsin-like protease may be recovered from the medium byconventional procedures including separating the cells from the mediumby centrifugation or filtration, if necessary after disruption of thecells, precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, e.g. ammonium sulphate, followed bypurification by a variety of chromatographic procedures, e.g. ionexchange chromatography, affinity chromatography, or the like.

Detergent Composition

According to the invention, the trypsin-like protease may typically be acomponent of a detergent composition. As such, it may be included in thedetergent composition in the form of a non-dusting granulate, astabilized liquid, or a protected enzyme. Non-dusting granulates may beproduced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452(both to Novo Industri A/S) and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000, ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in patent GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Other enzymestabilizers are well known in the art. Protected enzymes may be preparedaccording to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules, paste or liquid. A liquid detergent maybe aqueous, typically containing up to 70% water and 0-30% organicsolvent, or nonaqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as amylase, lipase, cutinase, another protease, cellulase,peroxidase and oxidase.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂ O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of e.g. the amide, imide, orsulfone type.

The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative as e.g. an aromatic borate ester,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners, orperfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. 7-12 such as 7-10.5.

Particular forms of detergent compositions within the scope of theinvention include:

1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                       7-12%                                                    (calculated as acid)                                                          alcohol ethoxysulfate                                                                              1-4%                                                     (e.g. C.sub.12-18  alcohol, 1-2 EO) or                                        alkyl sulfate (e.g. C.sub.18-18)                                              alcohol ethoxylate   5-9%                                                     (e.g. C.sub.14-15 alcohol, 7 EO)                                              sodium carbonate (as Na.sub.2 CO.sub.3)                                                            14-20%                                                   soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                        2-6%                                                     zeolite (as NaAlSiO.sub.4)                                                                         15-22%                                                   sodium sulfate (as Na.sub.2 SO.sub.4)                                                              0-6%                                                     sodium citrate/citric acid                                                                         0-15%                                                    (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)                sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                         11-18%                                                   TAED                 2-6%                                                     carboxymethylcellulose                                                                             0-2%                                                     polymers (e.g. maleic/acrylic acid                                                                 0-3%                                                     copolymer, PVP, PEG)                                                          enzymes              0-5%                                                     minor ingredients (e.g. suds                                                                       0-5%                                                     suppressors, perfume, optical                                                 brightener, photobleach)                                                      ______________________________________                                    

2) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                           6-11%                                                (calculated as acid)                                                          alcohol ethoxysulfate    1-3%                                                 (e.g. C.sub.12-18 alcohol, 1-2 EO)                                            or alkyl sulfate (e.g. C.sub.16-18)                                           alcohol ethoxylate       5-9%                                                 (e.g. C.sub.14-15 alcohol, 7 EO)                                              sodium carbonate (as Na.sub.2 CO.sub.3)                                                                15-21%                                               soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                            1-4%                                                 zeolite (as NaAlSiO.sub.4)                                                                             24-34%                                               sodium sulfate (as Na.sub.2 SO.sub.4)                                                                  4-10%                                                sodium citrate/citric acid                                                                             0-15%                                                (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)                carboxymethylcellulose   0-2%                                                 polymers (e.g. maleic/acrylic acid copolymer,                                                          1-6%                                                 PVP, PEG)                                                                     enzymes                  0-5%                                                 minor ingredients        0-5%                                                 (e.g. suds suppressors, perfume)                                              ______________________________________                                    

3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                           5-9%                                                 (calculated as acid)                                                          alcohol ethoxylate       7-14%                                                (e.g. C.sub.12-15  alcohol, 7 EO)                                             soap as fatty acid       1-3%                                                 (e.g. C.sub.16-22)                                                            sodium carbonate (as Na.sub.2 CO.sub.3)                                                                10-17%                                               soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                            3-9%                                                 zeolite (as NaAlSiO.sub.4)                                                                             23-33%                                               sodium sulfate (as Na.sub.2 SO.sub.4)                                                                  0-4%                                                 sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                             8-16%                                                TAED                     2-8%                                                 phosphonate (e.g. EDTMPA)                                                                              0-1%                                                 carboxymethylcellulose   0-2%                                                 polymers (e.g. maleic/acrylic acid copolymer,                                                          1-3%                                                 PVP PEG)                                                                      enzymes                  0-5%                                                 minor ingredients (e.g. suds suppressors,                                                              0-5%                                                 perfume, optical brightener)                                                  ______________________________________                                    

4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                           8-12%                                                (calculated as acid)                                                          alcohol ethoxylate       10-25%                                               (e.g. C.sub.12-15  alcohol, 7 EO)                                             sodium carbonate (as Na.sub.2 CO.sub.3)                                                                14-22%                                               soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                            1-5%                                                 zeolite (as NaAlSiO.sub.4)                                                                             25-35%                                               sodium sulfate (as Na.sub.2 SO.sub.4)                                                                  0-10%                                                carboxymethylcellulose   0-2%                                                 polymers (e.g. maleic/acrylic acid copolymer,                                                          1-3%                                                 PVP, PEG)                                                                     enzymes                  0-5%                                                 minor ingredients (e.g. suds suppressors                                                               0-5%                                                 perfume)                                                                      ______________________________________                                    

5) An aqueous liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                       15-21%                                                   (calculated as acid)                                                          alcohol ethoxylate   12-18%                                                   (e.g. C.sub.12-15 alcohol, 7 EO or                                            C.sub.12-15 alcohol, 5 EO)                                                    soap as fatty acid (e.g. oleic acid)                                                               3-13%                                                    alkenylsuccinic acid (C.sub.12-14)                                                                 0-13%                                                    aminoethanol         8-18%                                                    citric acid          2-8%                                                     phosphonate          0-3%                                                     polymers (e.g. PVP, PEG)                                                                           0-3%                                                     borate (as B.sub.4 O.sub.7)                                                                        0-2%                                                     ethanol              0-3%                                                     propylene glycol     8-14%                                                    enzymes              0-5%                                                     minor ingredients    0-5%                                                     (e.g. dispersants, suds suppressors,                                          perfume, optical brightener                                                   ______________________________________                                    

6) An aqueous structured liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                           15-21%                                               (calculated as acid)                                                          alcohol ethoxylate       3-9%                                                 (e.g. C.sub.12-15 alcohol, 7 EO                                               or C.sub.12-15 alcohol, 5 EO)                                                 soap as fatty acid (e.g. oleic acid)                                                                   3-10%                                                zeolite (as NaAlSiO.sub.4)                                                                             14-22%                                               potassium citrate        9-18%                                                borate (as B.sub.4 O.sub.7)                                                                            0-2%                                                 carboxymethylcellulose   0-2%                                                 polymers (e.g. PEG, PVP) 0-3%                                                 anchoring polymers as    0-3%                                                 e.g. lauryl methacrylate/acrylic acid copolymer;                              molar ratio 25:1; MW 3800                                                     glycerol                 0-5%                                                 enzymes                  0-5%                                                 minor ingredients        0-5%                                                 (e.g. dispersants, suds suppressors, perfume,                                 optical brighteners)                                                          ______________________________________                                    

7) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        fatty alcohol sulfate    5-10%                                                ethoxylated fatty acid monoethanolamide                                                                3-9%                                                 soap as fatty acid       0-3%                                                 sodium carbonate (as Na.sub.2 CO.sub.3)                                                                5-10%                                                soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                            1-4%                                                 zeolite (as NaAlSiO.sub.4)                                                                             20-40%                                               sodium sulfate (as Na.sub.2 SO.sub.4)                                                                  2-8%                                                 sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                             12-18%                                               TAED                     2-7%                                                 polymers (e.g. maleic/acrylic acid copolymer,                                                          1-5%                                                 PEG)                                                                          enzymes                  0-5%                                                 minor ingredients (e.g. optical brightener,                                                            0-5%                                                 suds suppressors, perfume)                                                    ______________________________________                                    

8) A detergent composition formulated as a granulate comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                         8-14%                                                  (calculated as acid)                                                          ethoxylated fatty acid monoethanolamide                                                              5-11%                                                  soap as fatty acid     0-3%                                                   sodium carbonate (as Na.sub.2 CO.sub.3)                                                              4-10%                                                  soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                          1-4%                                                   zeolite (as NaAlSiO.sub.4)                                                                           30-50%                                                 sodium sulfate (as Na.sub.2 SO.sub.4)                                                                3-11%                                                  sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                                                 5-12%                                                  polymers (e.g. PVP,    1-5%                                                   maleic/acrylic acid copolymer, PEG)                                           enzymes                0-5%                                                   minor ingredients (e.g. suds suppressors,                                                            0-5%                                                   perfume)                                                                      ______________________________________                                    

9) A detergent composition formulated as a granulate comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                         6-12%                                                  (calculated as acid)                                                          nonionic surfactant,   1-4%                                                   soap as fatty acid     2-6%                                                   sodium carbonate (as Na.sub.2 CO.sub.3)                                                              14-22%                                                 zeolite (as NaAlSiO.sub.4)                                                                           18-32%                                                 sodium sulfate (as Na.sub.2 SO.sub.4)                                                                5-20%                                                  sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                                                 3-8%                                                   sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                           4-9%                                                   bleach activator (e.g. NOBS or TAED)                                                                 1-5%                                                   carboxymethylcellulose 0-2%                                                   polymers (e.g. polycarboxylate or PEG)                                                               1-5%                                                   enzymes                0-5%                                                   minor ingredients      0-5%                                                   (e.g. optical brightener, perfume)                                            ______________________________________                                    

10) An aqueous liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                           15-23%                                               (calculated as acid)                                                          alcohol ethoxysulfate    8-15%                                                (e.g. C.sub.12-15 alcohol, 2-3 EO)                                            alcohol ethoxylate       3-9%                                                 (e.g. C.sub.12-15 alcohol, 7 EO                                               or C.sub.12-15 alcohol, 5 EO)                                                 soap as fatty acid (e.g. lauric acid)                                                                  0-3%                                                 aminoethanol             1-5%                                                 sodium citrate           5-10%                                                hydrotrope (e.g. sodium toluenesulfonate)                                                              2-6%                                                 borate (as B.sub.4 O.sub.7)                                                                            0-2%                                                 carboxymethylcellulose   0-1%                                                 ethanol                  1-3%                                                 propylene glycol         2-5%                                                 enzymes                  0-5%                                                 minor ingredients (e.g. polymers, dispersants,                                                         0-5%                                                 perfume, optical brighteners)                                                 ______________________________________                                    

11) An aqueous liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                         20-32%                                                 (calculated as acid)                                                          alcohol ethoxylate     6-12%                                                  (e.g. C.sub.12-15  alcohol, 7 EO                                              or C.sub.12-15  alcohol, 5 EO)                                                aminoethanol           2-6%                                                   citric acid            8-14%                                                  borate (as B.sub.4 O.sub.7)                                                                          1-3%                                                   polymer (e.g. maleic/acrylic acid copolymer,                                                         0-3%                                                   anchoring polymers as e.g.                                                    lauryl methacrylate/acrylic acid                                              copolymer and CMC)                                                            glycerol               3-8%                                                   enzymes                0-5%                                                   minor ingredients (e.g. hydrotropes,                                                                 0-5%                                                   dispersants, perfume, optical brighteners)                                    ______________________________________                                    

12) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        anionic surfactant (linear                                                                           25-40%                                                 alkylbenzenesulfonate, alkyl sulfate, alpha-                                  olefinsulfonate, alpha-sulfo fatty acid                                       methyl esters, alkanesulfonates, soap)                                        nonionic surfactant    1-10%                                                  (e.g. alcohol ethoxylate)                                                     sodium carbonate (as Na.sub.2 CO.sub.3)                                                              8-25%                                                  soluble silicates (as Na.sub.2 O, 2SiO.sub.2)                                                        5-15%                                                  sodium sulfate (as Na.sub.2 SO.sub.4)                                                                0-5%                                                   zeolite (as NaAlSiO.sub.4)                                                                           15-28%                                                 sodium perborate (as NaBO.sub.3.4H.sub.2 O)                                                          0-20%                                                  bleach activator (TAED or NOBS)                                                                      0-5%                                                   enzymes                0-5%                                                   minor ingredients      0-3%                                                   (e.g. perfume, optical brighteners)                                           ______________________________________                                    

13) Detergent formulations as described in 1)-12) where the content oflinear alkylbenzenesulfonate--or a part of it--is substituted by alkylsulfate (C₁₂ -C₁₈).

14) Detergent formulations as described in 1)-13) which contain astabilized or encapsulated peracid either as an additional component oras a substitute for already specified bleach systems.

15) Detergent compositions as described in 3), 7), 9) and 12) where thecontent of perborate is substituted with percarbonate.

16) Detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant as e.g. linear alkoxylatedprimary alcohol, a builder system (e.g. phosphate), enzyme and alkali.The detergent may also comprise anionic surfactant and/or a bleachsystem.

The trypsin-like protease of the invention may be incorporated inconcentrations conventionally employed in detergents. It is at presentcontemplated that, in the detergent composition of the invention, thetrypsin-like protease may be added in an amount corresponding to0.001-100 mg of the enzyme per liter of wash liquor.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the accompanying drawings, inwhich

FIG. 1 illustrates the construction of the expression plasmid pSX183further described in Example 3.

The present invention is further illustrated in the following exampleswhich should not, in any manner, be considered to limit the scope of thepresent invention.

MATERIALS AND METHODS

Strains

Aspergillus oryzae IFO 4177

Fusarium oxysporum DSM 2672 described in WO 89/06270

E. coli MC1000 (Cassabadan and Cohen, 1980)

Determination of trypsin-like Fusarium protease activity

The trypsin-like protease is assayed using the specific substrateN-Benzoyl-Larginine p-nitroanilide hydrochloride (L-BAPA or L-BAPNA).

Buffer

0.01M dimethylglutaric acid (Sigma D4379), 0.2M boric acid and 0.002Mcalcium chloride adjusted to pH 6.5 with NaOH.

Substrate

L-BAPA is available from Sigma (B3133) or Merck (Art.10754). A 0.2Mstock solution in dimethyl sulfoxide is prepared (87 mg/ml) (storefrozen) and diluted to 0.004M with the buffer described above.

Assay

The trypsin-like protease is diluted to approx. 15 μg/ml for the assay,100 μl of the diluted trypsin-like protease are mixed with 100 μl of0.004M L-BAPA in a 96 well microtiter plate. A blank with 100 μl bufferand 100 μl 0.004 M L-BAPA is used for correction.

The absorption change (delta OD/min) is monitored at 405 nm in an Elisareader for 10 minutes with readings every 5 minutes at 25° C. or roomtemperature.

The result is calculated relative to the trypsin content of a referenceFusarium trypsin-like protease.

Plasmids

pCDV1-PL (Noma et al., 1986)

p777 (described in EP 0 489 718)

pToC90 (described in WO 91/17243)

EXAMPLES EXAMPLE 1 Purification of the Trypsin-Like Protease fromFusarium

A crude F. oxysporum protease preparation was isolated from thesupernatant obtained by fermentation of the F. oxysporum DSM 2672 in asoy meal containing medium substantially as described in WO 89/06270.Subsequently, the Fusarium oxysporum trypsin-like protease was separatedfrom two other F. oxysporum proteases by 0-1M/0-25% sodiumchloride/isopropanol gradient elution at pH 7.0 from abacitracin-silicapolyol affinity column (Mortensen et al., 1989). Thetwo other proteases separated from the F. oxysporum trypsin-likeprotease by this procedure were, a subtilisin-like enzyme, and anaspartic protease with specificity required for the activation of theproform of the F. oxysporum trypsin-like protease (Asn-Ile cleavage).

The other purification steps were carried out in accordance withwell-known procedures, and included ion exchange chromatography onSeparose CL-6B (prior to the bacitracin separation), gelfiltration andconcentration by ultrafiltration.

The specific absorption for a 0.1% solution of the purified trypsin-likeF. oxysporum protease at 280 nm was determined to 1.32.

The purified protease was subsequently subjected to amino acidsequencing and analysis, and the N-terminal amino acid sequence wasdetermined.

EXAMPLE 2 Cloning of a Fusarium Oxysporum Trypsin Gene

mRNA preparation

Mycelium from a culture of the F. oxysporum strain DSM 2672 obtained asdescribed in Example 1 above, was crushed under liquid nitrogen,resuspended in 5M guanidine rhodanide, separated on a CsCl-sarcosylgradient, and mRNA was isolated therefrom. The mRNA was washed withethanol, and fractionated on an oligo-dT column (Chirgwin et al. (1978),Truelsen et al. (1979)). The quality of the resulting mRNA was tested inan in vitro translation assay using reticulocyte lysate and ³⁵S-methionine. An SDS-PAGE analysis showed bands from below 20 to morethan 100 kDa.

Preparation and screening of a cDNA library

A cDNA library of the mRNA was constructed in E. coli MC 1000 followingthe procedure of Noma et al. (1986). After transformation and platingabout 15000 colonies were replicated onto Whatman 540 filter paper. Amixed 17-mer probe based on the N-terminal of the enzyme was synthesised(T_(M) 42°-52° C.) ##STR1##

Split on the isoleucine codons the probe was divided into three pools of32 and three sets of filters were screened. After wash at 42° C. andre-isolation of possible candidates three positive clones wereidentified. These positive clones were all found with the ATC-isoleucineprobe.

Identification and analysis of the gene

Plasmids were prepared from the three clones according to Sambrook etal, 1989 and, following restriction mapping, two turned out to beidentical and the third was about 100 bp shorter. The longer clones weresequenced on both strands using the method of Maxam & Gilbert (1980) andthe resulting DNA sequence appears from SEQ ID No. 1.

The amino acid composition of the purified F. oxysporum trypsin-likeprotease obtained as described in Examples 1 was in accordance with thecomposition deduced from the DNA-sequence shown in SEQ ID No. 1.

Analysis of this sequence revealed a gene coding for a trypsin-likeprotease with a 17 amino acid signal peptide according to the rules ofvon Heijne (1986) (cleaved between Ala-17 and Ala-18) and a propeptideof 7 amino acids (Ala-Pro-Gln-Glu-Ile-Pro-Asn) between the signalpeptidase cleavage site and the known N-terminus of the enzyme. The roleof this hypothetical propeptide is unknown but proteases are normallymade as inactive zymogens.

The mature enzyme shows less than 50% identity with all known trypsinsbe it mammalian, from fruit fly, or bacterial (Streptomyces). Thehighest identity (48%) of the mature trypsin-like protease was foundwith a fruit fly trypsin.

Expression of the gene product

cDNA encoding the trypsin-like F. oxysporum protease, obtained asdescribed above, was inserted into the vector pCDV1-PL described by Nomaet al. (1986) resulting in the plasmid pSX180. The coding region of thecDNA was inserted as a Ncol-Xbal fragment into the Aspergillusexpression plasmid p777 (EP 0 489 718) which was cut with BamHI andpartially with Xbal. To join the 5'end of the cloned DNA to the vector asynthetic linker DNA KFN709/710 (illustrated in FIG. 1) was added to theligation reaction. The resulting plasmid pSX183 was co-transformed intoA. oryzae (IFO 4177) together with plasmid pToC90 carrying the amdS fromA. nidulans (WO 91/17243). Transformants were selected for growth onacetamide. When grown in YPD medium a protein appeared in thesupernatant slightly larger than the trypsin-like protease isolated fromF. oxysporum supernatants. Surprisingly no activity of the gene productcould be determined from hydrolysis of L-Benzoyl-Arginoyl-pNA (a trypsinsubstrate).

EXAMPLE 3 Activation of the Proenzyme

In an attempt to obtain an active trypsin-like protease, an aspartylprotease isolated from F. oxysporum supernatants (as described inExample 1) was added to the fermentation medium harbouring the inactivetrypsin-like protease.

By this treatment it was found that the protein from the transformed A.oryzae became the same size as the standard on an SDS gel and the sampleshowed tryptic activity as determined form hydrolysis ofL-Benzoyl-Arginoyl-pNA (data not shown). This indicates that the enzymeis expressed in the form of a proenzyme, and thus confirms the presenceof a propeptide as deduced from the DNA sequence. From the activitystudies it is apparent that the function of the propeptide is to keepthe enzyme in an inactive form.

REFERENCES CITED IN THE SPECIFICATION

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J.(1978). Isolation of biologically active ribonucleic acid from sourcesenriched in ribonuclease. Biochemistry 18:5294-5299.

Truelsen, E., Gausing, K., Jochimsen, B., Jorgensen, P., and Marcker, K.A. (1979). Cloning of soybean leghemoglobin structural gene sequencessynthesized in vitro. Nucleic Acids Res. 6:3061-3072.

Christensen, T., Woeldike, H., Boel, E., Mortensen, S. B., Hjortshoej,K., Thim, L., and Hansen, M. T. (1988). High level expression ofrecombinant genes in Aspergillus oryzae. Bio/Technology 6:1419-1422.

Maxam, A. M., and Gilbert, W. (1980). Sequencing end-labelled DNA withbase-specific chemical cleavages. Methods Enzymol. 65:499-

von Heijne, G. (1986). A new method for predicting signal sequencecleavage sites. Nucleic Acids Res. 14:4683-4690.

Hudson, L., and Hay, F. Practical Immunology, Third edition (1989),Blackwell Scientific Publications

Lipman and Pearson, Science 227, 1435 (1985)

Sambrook et at., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor, 1989

Saiki, R. K. et al., Science 239, 1988, pp. 487-491, 1988

Beaucage et at., Tetrahedron Letters 22, 1981, pp. 1859-1869

Matthes et al., The EMBO J. 3, 1984, pp. 801-805

Malardier et al., Gene 78 (1989), pp. 147-156

Zamost et al., Journal of Industrial Microbiology, Vol. 5, pp. 303-312,1990

Noma et al. (1986), Nature 319, 640-646

Cassabadan and Cohen (1980), J. Mol. Biol. 138, 179-203

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 998 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION:130..801                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION:58..801                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      ATCATCAACCACTCTTCACTCTTCAACTCTCCTCTCTTGGATATCTATCTCTTCACCATG60                GTCAAGTTCGCTTCCGTCGTTGCACTTGTTGCTCCCCTGGCTGCTGCCGCTCCTCAGGAG120               ATCCCCAACATTGTTGGTGGCACTTCTGCCAGCGCTGGCGACTTTCCC168                           IleValGlyGlyThrSerAlaSerAlaGlyAspPhePro                                       1510                                                                          TTCATCGTGAGCATTAGCCGCAACGGTGGCCCCTGGTGTGGAGGTTCT216                           PheIleValSerIleSerArgAsnGlyGlyProTrpCysGlyGlySer                              152025                                                                        CTCCTCAACGCCAACACCGTCTTGACTGCTGCCCACTGCGTTTCCGGA264                           LeuLeuAsnAlaAsnThrValLeuThrAlaAlaHisCysValSerGly                              30354045                                                                      TACGCTCAGAGCGGTTTCCAGATTCGTGCTGGCAGTCTGTCTCGCACT312                           TyrAlaGlnSerGlyPheGlnIleArgAlaGlySerLeuSerArgThr                              505560                                                                        TCTGGTGGTATTACCTCCTCGCTTTCCTCCGTCAGAGTTCACCCTAGC360                           SerGlyGlyIleThrSerSerLeuSerSerValArgValHisProSer                              657075                                                                        TACAGCGGAAACAACAACGATCTTGCTATTCTGAAGCTCTCTACTTCC408                           TyrSerGlyAsnAsnAsnAspLeuAlaIleLeuLysLeuSerThrSer                              808590                                                                        ATCCCCTCCGGCGGAAACATCGGCTATGCTCGCCTGGCTGCTTCCGGC456                           IleProSerGlyGlyAsnIleGlyTyrAlaArgLeuAlaAlaSerGly                              95100105                                                                      TCTGACCCTGTCGCTGGATCTTCTGCCACTGTTGCTGGCTGGGGCGCT504                           SerAspProValAlaGlySerSerAlaThrValAlaGlyTrpGlyAla                              110115120125                                                                  ACCTCTGAGGGCGGCAGCTCTACTCCCGTCAACCTTCTGAAGGTTACT552                           ThrSerGluGlyGlySerSerThrProValAsnLeuLeuLysValThr                              130135140                                                                     GTCCCTATCGTCTCTCGTGCTACCTGCCGAGCTCAGTACGGCACCTCC600                           ValProIleValSerArgAlaThrCysArgAlaGlnTyrGlyThrSer                              145150155                                                                     GCCATCACCAACCAGATGTTCTGTGCTGGTGTTTCTTCCGGTGGCAAG648                           AlaIleThrAsnGlnMetPheCysAlaGlyValSerSerGlyGlyLys                              160165170                                                                     GACTCTTGCCAGGGTGACAGCGGCGGCCCCATCGTCGACAGCTCCAAC696                           AspSerCysGlnGlyAspSerGlyGlyProIleValAspSerSerAsn                              175180185                                                                     ACTCTTATCGGTGCTGTCTCTTGGGGTAACGGATGTGCCCGACCCAAC744                           ThrLeuIleGlyAlaValSerTrpGlyAsnGlyCysAlaArgProAsn                              190195200205                                                                  TACTCTGGTGTCTATGCCAGCGTTGGTGCTCTCCGCTCTTTCATTGAC792                           TyrSerGlyValTyrAlaSerValGlyAlaLeuArgSerPheIleAsp                              210215220                                                                     ACCTATGCTTAAATACCTTGTTGGAAGCGTCGAGATGTTCCTTGAATAT841                          ThrTyrAla                                                                     TCTCTAGCTTGAGTCTTGGATACGAAACCTGTTTGAGAAATAGGTTTCAACGAGTTAAGA901               AGATATGAGTTGATTTCAGTTGGATCTTAGTCCTGGTTGCTCGTAATAGAGCAATCTAGA961               TAGCCCAAATTGAATATGAAATTTGATGAAAATATTC998                                      (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 224 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      IleValGlyGlyThrSerAlaSerAlaGlyAspPheProPheIleVal                              151015                                                                        SerIleSerArgAsnGlyGlyProTrpCysGlyGlySerLeuLeuAsn                              202530                                                                        AlaAsnThrValLeuThrAlaAlaHisCysValSerGlyTyrAlaGln                              354045                                                                        SerGlyPheGlnIleArgAlaGlySerLeuSerArgThrSerGlyGly                              505560                                                                        IleThrSerSerLeuSerSerValArgValHisProSerTyrSerGly                              65707580                                                                      AsnAsnAsnAspLeuAlaIleLeuLysLeuSerThrSerIleProSer                              859095                                                                        GlyGlyAsnIleGlyTyrAlaArgLeuAlaAlaSerGlySerAspPro                              100105110                                                                     ValAlaGlySerSerAlaThrValAlaGlyTrpGlyAlaThrSerGlu                              115120125                                                                     GlyGlySerSerThrProValAsnLeuLeuLysValThrValProIle                              130135140                                                                     ValSerArgAlaThrCysArgAlaGlnTyrGlyThrSerAlaIleThr                              145150155160                                                                  AsnGlnMetPheCysAlaGlyValSerSerGlyGlyLysAspSerCys                              165170175                                                                     GlnGlyAspSerGlyGlyProIleValAspSerSerAsnThrLeuIle                              180185190                                                                     GlyAlaValSerTrpGlyAsnGlyCysAlaArgProAsnTyrSerGly                              195200205                                                                     ValTyrAlaSerValGlyAlaLeuArgSerPheIleAspThrTyrAla                              210215220                                                                     __________________________________________________________________________

We claim:
 1. A DNA construct comprising the DNA sequence of SEQ ID NO:1.2. A DNA construct according to claim 1, wherein the DNA sequence is asshown in the appended SEQ ID No.
 1. 3. A recombinant expression vectorcomprising the DNA construct of claim
 1. 4. A cell comprising the DNAconstruct of claim
 1. 5. The cell of claim 4, wherein the cell is amicrobial cell.
 6. The cell of claim 5, wherein the cell is a bacterialcell or a fungal cell.
 7. The cell of claim 6, in which the bacterialcell is a cell of a gram-positive bacterium or a cell of a gram-negativebacterium and the fungal cell is a yeast cell or a cell of a filamentousfungus.
 8. A method of producing a recombinant protease enzyme, whereina cell containing the DNA construct of claim 1 is cultured underconditions conducive to the production of the protease, and the proteaseis subsequently recovered from the culture.
 9. The method of claim 8,wherein the recombinant protease enzyme is expressed in the form of aproenzyme and the cell is--cultured in the presence of a proteolyticenzyme capable of converting the proenzyme of the protease into a matureenzyme.
 10. The method of claim 8, wherein the proteolytic enzyme is ametallo-protease.
 11. A cell comprising the vector of claim
 3. 12. Thecell of claim 7, in which the gram-positive bacterium is Bacillus orStreptomyces, the gram-negative bacterium is Escherichia, the yeast cellis Saccharomyces, and the filamentous fungus is Aspergillus or Fusarium.13. The method of claim 10, wherein the proteolytic enzyme is a Bacillusmetallo-protease.